Download Nettargeted mutant mice develop a vascular phenotype and

The EMBO Journal Vol. 20 No. 18 pp. 5139±5152, 2001
Net-targeted mutant mice develop a vascular
phenotype and up-regulate egr-1
Abdelkader Ayadi1, Hong Zheng1,
Peter Sobieszczuk1,2, Gilles Buchwalter1,
Philippe Moerman3, Kari Alitalo4 and
Bohdan Wasylyk1,5
1
Institut de GeÂneÂtique et de Biologie MoleÂculaire et Cellulaire,
CNRS/INSERM/ULP, 1 Rue Laurent Fries, BP 163, 67404 Illkirch
cedex, France, 3Afdeling Morfologie en moleculaire pathologie,
Minderbroedersstraat 12, B-3000 Leuven, Belgium and 4Molecular/
Cancer Biology Laboratory, Haartmann Institute, University of
Helsinki, POB 21 (Haartmaninkatu 3), SF-00014 Helsinki, Finland
2
Present address: University of Manchester, School of Biological
Sciences, MTG±Incubator Building, Grafton Street,
Manchester M13 9XX, UK
5
Corresponding author
e-mail: [email protected]
The ternary complex factors (TCFs) Net, Elk-1 and
Sap-1 regulate immediate early genes through serum
response elements (SREs) in vitro, but, surprisingly,
their in vivo roles are unknown. Net is a repressor that
is expressed in sites of vasculogenesis during mouse
development. We have made gene-targeted mice that
express a hypomorphic mutant of Net, Netd, which
lacks the Ets DNA-binding domain. Strikingly, homozygous mutant mice develop a vascular defect and
up-regulate an immediate early gene implicated in
vascular disease, egr-1. They die after birth due to
respiratory failure, resulting from the accumulation
of chyle in the thoracic cage (chylothorax). The mice
have dilated lymphatic vessels (lymphangiectasis) as
early as E16.5. Interestingly, they express more egr-1
in heart and pulmonary arteries at E18.5. Net negatively regulates the egr-1 promoter and binds speci®cally to SRE-5. Egr-1 has been associated with
pathologies involving vascular stenosis (e.g. atherosclerosis), and here egr-1 dysfunction could possibly
be associated with obstructions that ultimately affect
the lymphatics. These results show that Net is involved
in vascular biology and egr-1 regulation in vivo.
Keywords: egr-1/Elk-3/ERP/Net/Sap-2
cellular responses to the activation of MAP kinase
pathways. Net differs from the other TCFs in that in
basal conditions, in which MAP kinases are not activated,
it strongly inhibits transcription. Repression is mediated
by two domains, the NID (Maira et al., 1996) and the
CID (Criqui-Filipe et al., 1999). The TCFs form ternary
complexes with SRF on serum response elements (SREs)
of immediate early gene promoters, such as c-fos, egr-1
and jun-B. The SRE is constitutively occupied by factors,
and extracellular signals are thought to lead to both
phosphorylation of the complex and changes in its
composition due to the exchange of TCFs.
The in vivo role of the TCFs is poorly understood. They
may regulate the expression of immediate early genes in
response to various inductive stimuli. The TCFs are
expressed in many cell types and tissues (Giovane et al.,
1994; Lopez et al., 1994; Magnaghi-Jaulin et al., 1996;
Nozaki et al., 1996; Sgambato et al., 1998), but their
precise in vivo expression patterns are not well known. Net
is expressed during mouse development at E7.5±8.5 in
developing vascular primordia, including the yolk sac
blood islands, allantoic vessels, heart endocardium and
dorsal aortae (Ayadi et al., 2001). Vascular endothelial
cell expression persists throughout development, raising
the possibility that Net may have functions during mouse
development in vasculogenesis and angiogenesis.
In order to study the function of Net in vivo, we
generated mice with a targeted disruption of the net gene.
Homozygous mutant mice are born with the expected
Mendelian frequency and appear normal. However, they
develop chylothorax with a high penetrance within the ®rst
postnatal weeks, and die of respiratory failure. The mice
have highly dilated thoracic lymphatic vessels, in which
net is expressed. They also have elevated levels of the
egr-1 mRNA in some vascular structures in the thoracic
region. Taken together, the results show that Net regulates
egr-1 and, in agreement with its expression pattern, has a
role in the vasculature.
Results
Introduction
The ternary complex factors (TCFs) form a subfamily of
Ets-domain transcription factors. The three TCFs, Elk-1,
Sap-1 and Net/Sap-2/Erp/Elk-3 (Price et al., 1996;
Wasylyk et al., 1998), have four conserved domains,
A±D. A is the Ets DNA-binding domain (DBD). The
B-box interacts with the serum response factor (SRF). C is
a transcriptional activation domain that is stimulated by
mitogen-activated protein (MAP) kinase phosphorylation.
The D-domain is a MAP kinase-binding site and a nuclear
localization signal. The TCFs are nuclear mediators of
ã European Molecular Biology Organization
Targeted disruption of the net gene
We deleted 1055 bp surrounding net exon 2, which
contains the ATG and codes for the ®rst 69 amino acids of
the DBD (Figure 1A). The targeting vector had, 5¢ to 3¢,
11.5 kb of genomic sequences, the PGK-NeoR cassette and
1.2 kb of genomic sequences. Recombinant embryonic
stem (ES) cell clones were injected into C57BL/6
blastocysts and chimeras were crossed to C57BL/6 and
129/Sv females. Net+/mutant mice were intercrossed to
generate homozygous mutant mice, as shown by Southern
blotting and PCR analysis of genomic DNA from the
offspring (Figure 1B and C).
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A.Ayadi et al.
Fig. 1. Targeted mutagenesis of the net gene. (A) Schemes. Exon 2 contains the translation initiation codon and encodes amino acids 1±69 of Net. The
3¢ probe for Southern blots, the 13 (wild-type) and 5 (mutant) kb XbaI fragments and the PCR primers (uc54, uc56 and uc57) are shown. B, BamHI;
X, XbaI. (B) Southern analysis of XbaI-digested DNA from the progeny from a heterozygous (wild-type/mutant) inter-cross. The 3¢ probe reveals 13 kb
wild-type (WT) and 5 kb targeted (M) alleles. (C) PCR analysis of the same progeny. The products for the wild-type (WT, 1550 bp) and the targeted
(M,1300 bp) alleles are indicated. (D) Analysis of net transcripts by RT±PCR. RNA from E16.5 wild-type and homozygous mutant embryos was used
for RT±PCR with primers from exons 1±4. As expected, mutant RNA is not ampli®ed with exon 2 primers due to the deletion, but is ampli®ed with
exon 3 + 4 primers. Exon 1 + 3 (ex1/ex3 set) primers produce a shorter product (90 bp) with the mutant as opposed to the wild-type (297 bp) RNA.
The deduced mutant Net mRNA, Netd, is shown. The in-frame ATG is indicated in italics. (E) Western blots of lung extracts from 2-week-old wildtype, heterozygous and homozygous mice. The #375 antibody, raised against a peptide encoded by exon 3 (Giovane et al., 1994, 1997), detects a
47 000 Da protein in wild-type (+/+) and heterozygous (+/d) samples. The heterozygous (+/d) and homozygous (d/d) samples have a 37 000 Da band,
Netd, as expected from initiation at the internal ATG. (F) Basal c-fos SRE reporter activity in transfected MEFs generated from wild-type and
homozygous mutant (netd/d) embryos. The histogram shows the average absolute luciferase activity for three experiments repeated in triplicate.
Characterization of the Net mutant mice
Net mRNA was examined by RT±PCR with primers that
amplify across the junctions between exons 1 and 4
(Figure 1D). The expected products were obtained with
wild-type and homozygous mutant mice and primers
between exons 1 and 2, 2 and 3, and 3 and 4. With primers
between 1 and 3, the expected 297 bp fragment was
obtained from wild-type mice, and a shorter 90 bp
fragment from the homozygous mutants, corresponding
to splicing between exons 1 and 3, as shown by sequencing
the PCR product (data not shown). The exon 1±3 splice
product was not detected in wild-type mice, suggesting
that it is speci®c for the mutant. All the net transcripts
detected by northern blotting were found to be reduced in
size, as expected from deletion of exon 2 (data not shown).
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The new transcript contains an in-frame AUG in exon 3
(nucleotide 571, amino acid 93) that is expected to be
a good translation initiation site since there is an A
at ±3 and a G at +4 (Kozak, 1991; Giovane et al., 1994).
The predicted 37 000 Da Net protein was detected in
heterozygous and homozygous mice, by western blotting
with two different antibodies against Net, PAb 375
(Figure 1E) and PAb 376 (data not shown). As expected,
the wild-type 47 000 Da Net protein was not detected in
the homozygous mutant mice. The mutant Net protein,
Netd, lacks the Ets domain (amino acids 1±89), which is
encoded by sequences upstream from the new start (M93).
The loss of the DBD is expected to inhibit DNA binding
and thereby decrease repression, measured with a c-fos
SRE in a reporter under basal conditions as described
Net negatively regulates egr-1 in vivo
129Sv background, with ~80% of the netd/d mice dying
within 6 weeks (Figure 2B). The same phenotype was
observed following 10 back-crosses with 129Sv mice, and
12 back-crosses with C57BL/6 mice, showing that the
phenotype was not strictly dependent on the background,
and was tightly linked to the mutated allele. The homozygous mice developed signs of respiratory distress,
became physically inactive and died within ~2 days.
netd/d animals with respiratory distress were found to have
a milky liquid in the thoracic cavity (Figure 2C), which
was shown to be chyle, based on its high level of
triglycerides (18.4 mmol/l versus 1.5 mmol/l in the
plasma), and a high proportion of lymphocytes (80% of
the cells by FACS analysis; data not shown). The chylous
effusion ®lled the pleural space and compressed the lungs,
as shown by examination of paraf®n sections of the thorax
(Figure 3A and B). The survival of netd/d mice with
respiratory distress could be extended for a few days, by
draining the chylous effusion by thoracocentesis using a
syringe. These results indicate that the netd/d mice die from
respiratory failure due to compression of the lungs by
chyle, in other words from `congenital' chylothorax
(Merrigan et al., 1997; de Beer et al., 2000).
Dilated lymphatic vessels in the netd/d mice
Fig. 2. The phenotype of netd/d mice. (A) Genotype frequency of liveborn animals from a heterozygous inter-cross. (B) Decreased survival
of netd/d mice compared with +/+ and +/d animals. (C) Typical
phenotype developed by netd/d mice. This animal had respiratory
distress at 6 days and died 2 days later. The thoracic cavity is full
of chyle.
previously (Giovane et al., 1994). SRE activity was
measured by transfection of an SRE-luciferase reporter in
primary mouse embryo ®broblasts (MEFs) derived from
mutant and normal mice. As expected, SRE activity was
four times higher in the netd/d compared with wild-type
®broblasts under basal conditions (Figure 1F).
Netd/d mice die postnatally of chylothorax
Cross-breeding of heterozygous net+/d mice (from both
mixed 129Sv/C57BL/6 and pure inbred 129Sv genetic
backgrounds) gave viable homozygous offspring with the
expected Mendelian ratios (Figure 2A), indicating that
netd/d mice develop normally. The netd/d mice began to die
within a few weeks. The death rate was highest on the
Highly dilated vessels were observed in histological
sections of the thoracic wall of the netd/d mutant mice
(Figure 3C and D). These appeared to be lymphatic
vessels, on the basis of their extremely thin endothelial
lining and the absence of luminal red blood cells. Vascular
endothelial growth factor receptor 3 (VEGFR-3) knock-in
mice, which speci®cally express LacZ in the lymphatics
(Dumont et al., 1998), were used to con®rm the identity of
the dilated vessels. The VEGF-R3+/±netd/d mice, which
were generated by crossing compound mutant mice (vegfr3+/±net+/d), were phenotypically indistinguishable from
the netd/d mice, indicating that the lack of one VEGFR-3
allele did not enhance the effect of the net mutation. The
X-gal-stained blue lymphatic vessels inside the thoracic
wall were very dilated in the netd/d animals with a
developing chylothorax compared with their net+/+ littermates (Figure 4A and B). In contrast, the lymphatic vessels
of the pericardium and skin were similar (Figure 4C±F),
showing that the dilation was speci®c for the chest wall.
The increase in the diameter of the thoracic lymphatics in
the netd/d mice was observed after birth (Figure 4G and H),
and even at E16.5 (data not shown), well before the onset
of the respiratory distress and chylothorax.
Net mRNA is expressed in the lymphatic
endothelium
We have found that net mRNA expression is highly
restricted to endothelial cells during mouse development
(Ayadi et al., 2001). We studied whether net is expressed
in the lymphatic vessels by in situ hybridization (ISH),
using VEGFR-3 as a speci®c marker (Kaipainen et al.,
1995). Using serial frozen sections from E16.5 embryos,
net and VEGFR-3 expression were found to be colocalized in the thoracic duct (Figure 5A±C), as well as in
the intestinal (Figure 5D±F) and skin (data not shown)
lymphatics. However, net expression is wider than
endothelial cells, especially in the thoracic duct, where it
is also detected within the muscle layer (Figure 5B; Ayadi
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Fig. 3. Histology of the thorax. Haematoxylin±eosin-stained cross-sections of wild-type (A) and mutant (B) thoraxes are shown. Bars = 0.8 mm. The
netd/d mouse was killed at 8 days, when it developed respiratory distress symptoms, together with a net+/+ littermate control. The pleural space is
expanded in the mutant, ®lled by the chylous effusion (asterisk), which has compressed the lungs and the heart. (C and D) Magni®cations of the
dashed squares in (A) and (B), respectively. The netd/d thoracic wall (D) has dilated lymphatic vessels compared with the control (C). R, ribs; lv,
lymphatic vessel. Bars = 0.2 mm.
et al., 2001). net was found to have a vascular pattern of
expression on the thoracic wall by whole-mount RNA
hybridization (Figure 5G and H). VEGFR-3 was expressed
in a similar pattern, as shown by X-gal staining of the
VEGF-R3+/± mice (Figure 5I). These results show that net
is expressed in the lymphatic vessels, which are affected in
the netd/d animals.
Net mutation leads to localized up-regulation of
egr-1 expression
We studied the molecular basis of the phenotype of the
netd/d mice by RNA ISH of E16.5 stage embryos. We
initially used endothelial markers, because lymphatics
have a venous origin (Kaipainen et al., 1995) and net is
expressed in endothelial cells. We chose genes with
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functional Ets-binding sites in their regulatory regions,
including Tie1, Tie2 and VEGFR-1 (Flt-1) (Wakiya et al.,
1996; Schlaeger et al., 1997; Iljin et al., 1999). However,
no obvious differences were found (data not shown and
Figure 7E and F). We also studied c-fos and egr-1, two
SRE-containing genes that are regulated by the TCFs
(reviewed in Treisman, 1994; Wasylyk et al., 1998) and
are expressed in the perichondrium of bones (Dony and
Gruss, 1988; Sandberg et al., 1988; McMahon et al.,
1990), similarly to net (Ayadi et al., 2001). The expression
levels of c-fos or egr-1 in bony structures, such as in the
perichondrial interface of basioccipital bone and vertebrae
(Figure 6A±F) and the articular joint from the hind limb
(Figure 6G±J), were similar in wild-type and net mutant
mice. However, increased expression of egr-1 was
Net negatively regulates egr-1 in vivo
regulation was no longer detected in the heart, but there
was a striking increase in egr-1 expression in one or
several major pulmonary arteries of the mutant embryos
(Figure 8). egr-1 up-regulation was restricted to a
particular area of the lung, as shown with different sagittal
sections (Figure 8C, F and I). The egr-1 signal was homogeneous throughout the vascular wall in the mutant
embryos, compared with the heterogeneous labelling in
the equivalent blood vessel in the littermates (Figure 8J±L).
We also studied egr-1 expression in thoracic tissues from
mutant mice after birth, but before the onset of chylothorax
(Figure 9). There was a widespread increase in egr-1
expression in the lungs of netd/d mice (Figure 9C and D;
arrowheads), which upon closer examination (Figure 9G
and H) was found to be patchy compared with the more
homogeneous background staining in the wild-type mice,
and located in particular in blood vessels as well as other
unidenti®ed cells in the lung parenchyma (arrows). In
conclusion, net mutation results in egr-1 up-regulation in
restricted parts of the vasculature, in the atrial wall of the
heart at E16.5, in pulmonary arteries at E18.5 and in lung
arteries after birth.
Net negatively regulates the egr-1 promoter
Fig. 4. Dilatation of the thoracic lymphatic vessels in netd/d mice. The
LacZ-VEGFR-3 reporter strain was used to identify lymphatic vessels
(Dumont et al., 1998). The lymphatic vessels of net+/+VEGFR-3+/± (A,
C, E and G) and netd/dVEGF-R3+/± mice (B, D, F and H) were stained
with X-gal (blue). (B, D and F) Lymphatic staining of a netd/d mouse
that developed chylothorax at 10 days of age. Lymphatics of the
mutant thoracic wall (B) are dilated compared with the +/+
littermate (A). (C and D) Pericardial and (E and F) chest skin
lymphatic vessels. Bars = 42 mm. (G and H) Thorax from a 5-day-old
netd/d mouse before pleural effusion and a net+/+ littermate. R, ribs;
ic, intercostal region. Bars = 90 mm.
observed in the atrium wall of the heart of netd/d embryos
(Figure 7A±D). Surprisingly, there was no increase in
adjacent tissues, such as the aorta and the perichondrium
of the ribs. Although net was expressed in the endocardium, in the endothelial cells (compare with the VEGFR-1
marker, Figure 7E and F), the perichondrium of the ribs
and the cartilaginous condensation of the trachea, its
pattern of expression was similar in the wild-type and
mutant mice (Figure 7G and H). At E18.5, egr-1 up-
In transfection assays, the c-fos SRE is negatively
regulated by Net under basal conditions (Giovane et al.,
1994), raising the possibility that the egr-1 promoter is
also negatively regulated by Net. The egr-1 promoter
contains ®ve SREs, of which the three upstream elements
(3±5, Figure 10A) are critical for the induction of egr-1
promoter activity (Clarkson et al., 1999). We investigated
whether Net negatively regulates egr-1 promoter activity,
using net antisense constructs to down-regulate Net
(Giovane et al., 1994). Mouse NIH-3T3 ®broblasts were
transfected with the egr-1200-Luc reporter (Clarkson et al.,
1999) and increasing quantities of the antisense net
plasmid. Down-regulation of Net reproducibly increased
egr-1 promoter activity (Figure 10B). The extent of upregulation is signi®cant, since it is quantitatively similar to
the effect of co-expression of Sap-1a and Elk-1 (Clarkson
et al., 1999). The antisense is speci®c, since it downregulates endogenous Net without affecting the levels of
the closely related protein Elk-1 (Giovane et al., 1994 and
data not shown). Deletion of the three critical upstream
SREs abolished the stimulation resulting from Net
down-regulation (compare egr-1200- and egr-250-Luc,
Figure 10B). These results show that the egr-1 promoter is
negatively regulated by Net.
Net is recruited to the c-fos SRE by SRF to form a
ternary complex, but can bind alone to consensus Ets
motifs (Giovane et al., 1994; Maira et al., 1996). SAP-1a
and Elk-1 have been shown to bind autonomously to the
Ets motif of egr-1 SRE-5 and to form ternary complexes
with SRF on this element (Clarkson et al., 1999). We
investigated whether Net has similar properties, using
bandshift experiments and SRE-5 probes. We found that
Net forms a complex with the wild-type probe in the
absence of SRF (Figure 10C, lanes 1 and 2). Complex
formation is prevented by mutation of the Ets motif (mut
ets, lanes 5 and 6) but is not inhibited by mutation of the
SRF motif (mut SRF, lanes 9 and 10). SRF forms a
complex in an SRF motif-dependent manner (lanes 3, 7
and 11), and SRF + Net form a ternary complex when both
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Fig. 5. Co-expression of net and VEGFR-3 RNAs in E16.5 embryos. (A±F) ISH of sagittal sections with 35S-labelled riboprobes. The signal grains are
white dots on a dark ®eld. (A and D) Bright-®eld sections are shown for histology. (B and C) Net and the VEGFR-3 lymphatic marker are expressed
in the thoracic duct (arrows). (E and F) Net is also expressed in lymphatic (arrows) and blood vessels in the gut. (G±I) Whole-mount ISH of thoracic
biopsies. The speci®c signal has a dark blue colour. (G) Net is expressed in a plexus pattern (arrow). (H) The higher magni®cation shows Net staining
of a lymphatic vessel (arrowhead). (I) Same magni®cation as (H), to show the aspect of lymphatics (arrowhead) in the thoracic wall stained with the
VEGFR-3 probe. Ao, aorta; Oe, oesophagus; r, ribs; St, sternum; Td, thoracic duct; Tr, trachea; Ve, vertebrae. Bars = 50 mm (A±F), 200 mm (G),
100 mm (H and I).
motifs are intact (TC, lanes 4, 8 and 12). We compared the
rate of dissociation of the ternary (TC) and binary Net
complexes in the presence of excess cold SRE-5 probe
(Figure 10D). The ternary complex dissociates more
slowly, showing that it is more stable. These results
show that Net can bind autonomously and form ternary
complexes with SRF on SRE-5.
We investigated whether deletion of the Ets domain in
Netd inhibits binding and recruitment by SRF to SRE-5.
Using in vitro translated proteins, we found that Netd was
unable to form either autonomous or ternary complexes
with SRE-5 (Figure 10E, compare lanes 5 and 6 with 1±4).
Using lung nuclear extracts, we detected several complexes that are sensitive to mutation of the Ets motif, B1,
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B3 and B4 (Figure 10F, the part of the gel with differences
is shown). B1 correspond to ternary complexes, since they
are supershifted by SRF antibodies (not shown), migrate
above the SRF complex (B2), have a similar migration as
the ternary complex formed with in vitro translated
proteins and are inhibited by mutation of the Ets motif.
B2 is the SRF complex, since it is supershifted with SRF
antibodies, inhibited by mutation of the SRF motif and comigrates with the in vitro complex (data not shown). B3 is
not signi®cantly affected by Net antibodies, and does not
co-migrate with in vitro complexes, suggesting that it is
formed by other Ets proteins. B4 co-migrates with in vitro
Net complexes and its intensity is reduced by Net
antibodies, suggesting that it is composed partly of Net
Net negatively regulates egr-1 in vivo
Fig. 6. Unaltered expression of c-fos and egr-1 RNA in bony structures of netd/d E16.5 embryos. ISH of net+/+ (A, C, E, G and I) and netd/d (B,
D, F, H and J) embryos. (C and E), (D and F), (I) and (J) are the dark ®elds corresponding to the bright ®elds (A), (B), (G) and (H), respectively.
Expression of: (i) c-fos (C and D) and egr-1 (E and F) is not altered in the mutant embryo in the articular surface between the basioccipital bone and
vertebrae (arrowheads); and (ii) of egr-1 (I and J) in the articular joint space from the hind limb (arrowheads). Ar, articular space; Bo, basioccipital
bone; Br, brain; Hu, humerus; OC, otic capsule; Ra, radius; Ve, vertebrae. Bars = 90 mm (A±F) and 110 mm (G±J).
complexes. The B4 complex in netd/d extracts has a lower
intensity and is not affected by Net antibodies (lanes 2, 3, 5
and 6), suggesting that it contains other Ets proteins. These
results show that deletion of the Ets domain in Netd
inhibits binding to SRE-5 in the presence or absence of
SRF.
Discussion
We produced Net mutant mice lacking exon 2, which
contains the translation initiation codon and codes for most
of the Ets DBD (Giovane et al., 1994, 1997). The mutant
mice express a mRNA isoform that lacks exon 2 due to
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Fig. 7. Up-regulation of egr-1 RNA in the heart of netd/d E16.5 embryos. ISH on sagittal frozen sections of the thoracic region. (A and B) Bright
®elds, (C±H) corresponding dark ®elds for net+/+ (A, C, E and G) and netd/d (B, D, F and H) mice. (C and D) Egr-1 labelling is stronger in the atrial
wall of the netd/d heart (arrowheads) but not, for example, in the ribs or the aorta. (E and F) VEGFR-1 endothelial marker. (G and H) Net expression.
VEGFR-1 and net are expressed in the atrial wall (arrowheads, E±H). Ao, aorta; At, atrium; Ht, heart; Lu, lung; Ri, ribs; Th, thymus; Tr, trachea. Bar
= 110 mm.
splicing between exons 1 and 3, and in which all the other
exons are spliced normally (Figure 1D and data not
shown). This RNA has an in-frame ATG translation
initiation codon with an optimal Kozak sequence. It codes
for a shorter protein, Netd, which lacks the Ets DBD. The
net gene produces several alternatively spliced mRNAs,
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including net-b and net-c (Giovane et al., 1997 and data
not shown). We cannot exclude the possibility that the
exon 1±3 splice occurs naturally, even though we did not
detect it in several mouse tissues (data not shown). Netd
lacks the Ets domain and consequently does not bind to
DNA in vitro. However, we cannot exclude the possibility
Net negatively regulates egr-1 in vivo
Fig. 8. Spatially restricted up-regulation of egr-1 RNA in the lung vasculature of netd/d E18.5 embryos. Egr-1 ISH on sagittal sections from the
thoracic cage of net+/+ (B, E, H and K) and netd/d (C, F, I and L) embryos. The bright ®elds (A, D, G and J) of the net+/+ sections are shown for
histology. Comparable sections (1±3) are shown for net+/+ and netd/d embryos. In section 1, egr-1 expression is similar in the wild-type and mutant
mice, whereas in the other two sections there is stronger labelling in individual pulmonary arteries in the mutant (arrowheads; F and I). There is no
difference in egr-1 labelling in other sites of expression, such as the thymus, the rib perichondrium and the walls of the vena cava as well as other
vascular structures. (J, K and L) Higher magni®cation of sections 2, showing egr-1 up-regulation in the wall of an artery of a netd/d embryo (L,
arrowheads), compared with the same vessel of a wild-type embryo (K, arrowheads), where egr-1 is detected within a restricted area. Ar, artery;
Ht, heart; L, vascular lumen; Li, liver; Lu, lung; Ri, ribs; Th, thymus; VC, vena cava. Bars = 200 mm (A±I) and 25 mm (J±L).
that Net is recruited to some promoters by protein±protein
interactions in vivo, or has DNA binding-independent
functions. Netd retains a number of functional domains,
including the activation domain (C) and interaction
domains with SRF (B), E47 (NID), CtBP (CID) and
MAP kinases (D, JEX) (Maira et al., 1996; Criqui-Filipe
et al., 1999; Ducret et al., 2000). A brain-speci®c isoform
of Elk-1, sELK-1, that lacks the ®rst 54 amino acids of the
DBD has been described recently. This deletion severely
compromises but does not abolish ternary complex
formation on the SRE in vitro. sElk-1 plays an opposite
role to Elk-1 in nerve growth factor-driven PC12 neuronal
differentiation (Vanhoutte et al., 2001). As Net is a
repressor under basal conditions (Giovane et al., 1994), the
loss of repressor function by the mutant protein should
increase the activity of cellular SRE elements. As
expected, we found that c-fos SRE activity is enhanced
in netd/d MEFs transfected with an SRE reporter.
Furthermore, Netd cannot bind to DNA by itself or
together with SRF. In netd/d MEFs, egr-1 levels are higher
under basal conditions and somewhat lower following
induction by ®broblast growth factor-2 (FGF-2), consistent with the loss of a repressor that is converted to an
activator by FGF-2-induced signalling (data not shown).
These data strongly suggest that Netd is a hypomorphic
mutant that has a weaker effect than the corresponding
wild-type protein. It is unlikely that the protein has a transdominant effect, since heterozygous animals are phenotypically identical to their wild-type littermates. Netd is
expressed in amounts similar to the wild-type protein. In
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Fig. 9. Egr-1 ISH of lungs before the onset of chylothorax. Equivalent sagittal frozen sections from the thoracic region of 2-day-old net+/+ (A, C, E
and G) and netd/d (B, D, F and H) mice. (C and D) Lower magni®cation showing a speci®c increase in the egr-1 signal throughout netd/d lungs
(arrowheads). (E±H) Higher magni®cations of similar regions of the lungs. (G and H) Patchy egr-1 up-regulation is detected in pulmonary blood
vessels (arrowheads) and parenchyma (arrows). Ht, heart; Lu, lung; Th, thymus; Ri, ribs; VC, vena cava. Bars = 150 mm (A±D) and 50 mm (E±H).
addition, inhibition by a dominant-negative Net protein
of the other TCFs, which are positive regulators, would
decrease rather than increase the activity of the SRE
(Vanhoutte et al., 2001).
Homozygous netd/d mutant animals develop congenital
chylothorax, which has not been reported previously for
mice. Chylothorax is a rare condition found in humans and
other animals that results from accumulation of chyle in
5148
the pleural space due to disruption of the thoracic duct or
one of its collaterals (Merrigan et al., 1997; de Beer et al.,
2000). Such rupture can result from identi®able causes,
such as trauma, infection or neoplasia, or can arise
spontaneously neonatally or during infancy, most often
due to congenital defects in the circulation (van Straaten
et al., 1993; de Beer et al., 2000). The absence of
communication between the lymphatics or obstruction of
Net negatively regulates egr-1 in vivo
Fig. 10. Net represses the activity of and binds to the egr-1 promoter. (A) Schemes of the mouse egr-1 promoter±luciferase reporters. Egr-1200-Luc
contains 1200 bp of the egr-1 promoter with its ®ve SREs (closed squares), whereas egr-250-Luc contains 250 bp upstream from the transcription start
(arrows) with two proximal SREs. (B) Decreasing endogenous Net expression stimulates the egr-1 reporter through the distal SREs. NIH-3T3 cells
were transfected in triplicate with egr-1 reporters and the p601D-anti-net plasmid that produces antisense net RNA, and luciferase activity was
measured. An increasing amount of anti-net leads to signi®cant and reproducible activation of the egr-1200-Luc reporter, but not egr-250-Luc. (C, D
and E) Equal amounts of in vitro translated Net, Netd and SRF proteins were used for gel retardation assays with wild-type or mutant SRE-5 probes
(mut ets and mut SRF: mutated binding sites). Proteins and SRE-5 probes were incubated as indicated at the top and complexes were resolved by
PAGE. Arrows indicate the complexes formed by Net, SRF and both proteins (TC, ternary complex), as well as a non-speci®c complex (NC) and the
free probe (FP). (D) To compare off-rates, complexes were allowed to form for 25 min at 25°C and, after cooling on ice, a 500-fold excess of cold
probe was added, the reactions were incubated for the indicated times at 0°C and then immediately run on the gel. 30¢* was incubated for 30 min
without competitor. (F) Nuclear extracts (Giovane et al., 1997) from net+/+ and netd/d lungs and in vitro translated Net and SRF proteins were used
simultaneously for gel retardation assays with wild-type or mut ets SRE-5 probes. The Net antibodies were #375 (Giovane et al., 1994).
lymphatic ¯ow is thought to cause back pressure, dilation
(lymphangiectasis) and leakage of lymph. netd/d mice have
extremely dilated lymphatic vessels speci®cally in the
thoracic wall. Dilation precedes the onset of chylothorax,
raising the possibility that there is a defect that alters
lymphatic drainage. Net mRNA is expressed in lymphatic
endothelium, including the thoracic duct and lymphatic
vessels in the thoracic wall, suggesting that Net could be
involved in the formation of the lymphatic network.
However, we did not detect any obvious structural
abnormalities in thoracic vessels or surrounding tissues
by electron microscopy (data not shown), suggesting
either that the primary defect is subtle or that it is located
elsewhere. Interestingly, a9 integrin `knock-out' mice
have recently been reported to develop chylothorax
(Huang et al., 2000). However, the phenotype of netd/d
differs from that of a9±/± in that: (i) it is not fully penetrant
and the time of onset is later; (ii) it shows no signs of
oedema and in¯ammation surrounding the lymphatics
(Figure 3D and data not shown); and (iii) it exhibits
lymphangiectasis, which is not observed in a9±/± mice.
These differences suggest that the underlying defects are
different. The causes of congenital chylothorax in humans
are unknown. Our results raise the possibility that net gene
mutation could potentially be a genetic cause of congenital
chylothorax. Interestingly, the net gene is located on
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A.Ayadi et al.
chromosome 12q, and partial trisomy of 12q has been
detected in one case of chylothorax (Houf¯in et al., 1993).
However, additional loci may be involved, since trisomy
21 has also been associated with congenital chylothorax
(Hamada et al., 1992).
We have found that netd/d mice express more egr-1
mRNA. Transcription of egr-1 is induced rapidly and
transiently by a variety of mitogens in most cell lines
without de novo protein synthesis, and with a pattern
similar to c-fos (reviewed by Gashler and Sukhatme, 1995;
Liu et al., 1998). The similarity to c-fos may be due to
the SRE regulatory elements (Treisman, 1992). In vitro
studies have shown that the TCFs Net, Sap1 and Elk1 are
recruited to SREs by a dimer of SRF (Treisman, 1994;
Wasylyk et al., 1998). Net is able to down-regulate an
isolated c-fos SRE in transient transfection assays
(Giovane et al., 1994; this study). However, c-fos expression is not altered in netd/d animals or MEFs (data not
shown). This may indicate that Net does not regulate c-fos
expression in vivo (at least where studied), or that
redundancy amongst the TCFs, or the nature of the Netd
mutation, mask its contribution. The egr-1 promoter has
®ve SREs that mediate its response to various stimuli
(McMahon and Monroe, 1995; Clarkson et al., 1999;
Santiago et al., 1999a). Therefore, Egr-1 can be expected
to be more sensitive to inactivation of TCFs (Arsenian
et al., 1998). Our results provide the ®rst in vivo evidence
for regulation of egr-1 by a TCF.
The striking feature of egr-1 up-regulation in netd/d mice
is its speci®city. At E16.5, egr-1 expression is increased in
the atrial wall of the heart, at E18.5 in the vascular wall of
some major pulmonary arteries, and after birth in the lung,
in particular around blood vessels. egr-1 expression is
not altered overtly in other sites where it is expressed,
including perichondrial tissues, the thymus, and MEFs in
response to certain inducers (data not shown). Recently,
Egr-1 protein has been shown to be expressed in a variety
of cells in adult mice, including hepatocytes, neuronal
cells, cardiomyocytes, and endothelial and vascular
smooth muscle cells (Tsai et al., 2000). The authors
point out that Egr-1 expression is heterogeneous, raising
the possibility that only a subset of endothelial cells or
cardiomyocytes express Egr-1. These cells could be those
that overexpress egr-1 in the net mutant mice.
Increased egr-1 expression could be a direct consequence of Net mutation. Net is expressed speci®cally in a
number of cell types, including cardiomyocytes, endothelial and vascular smooth muscle cells (Ayadi et al.,
2001; Figure 7G and H). Furthermore, Net binds to the
egr-1 SRE and the endogenous protein represses egr-1
promoter activity. These results are consistent with direct
regulation. However, Net mutation does not increase egr-1
expression in all the cells in which they are co-expressed.
Furthermore, increased egr-1 expression is not restricted
to thoracic lymphatic vessels, the ultimate location of the
phenotype, showing that the phenotype cannot be
explained simply by egr-1 up-regulation. There are
various potential explanations as to why up-regulation is
restricted to some cells. The TCFs regulate SRE activity in
response to particular signals. The cells with increased
egr-1 expression could be responding to speci®c signals
that are regulated negatively by Net. Alternatively, the
netd/d mutation may be hypomorphic, and only the most
5150
sensitive pathways may be affected by this mutation.
Nevertheless, this is the ®rst in vivo evidence for the
regulation of an immediate early gene by a TCF.
It is possible that the chylothorax phenotype and egr-1
up-regulation are linked. There is evidence that Egr-1 is
important in vascular biology, especially in pathological situations (reviewed in Khachigian and Collins,
1997; Silverman and Collins, 1999). Following vascular
injury, egr-1 expression is highly induced in endothelial and vascular smooth muscle cells at wound
margins (Khachigian et al., 1996; Morawietz et al.,
1999; Santiago et al., 1999b,c). Inducible egr-1 expression
may coordinate the expression of multiple target genes
involved in cell movement and replication in blood vessel
walls. Ultimately, these cellular changes can result in the
pathogenesis of vascular occlusive lesions such as
restenosis or atherosclerosis. Egr-1 has been implicated
directly in the migration and proliferation of smooth
muscle cells, using antisense and DNA-targeted enzyme
approaches (Santiago et al., 1999b,c). netd/d mice overexpress egr-1 in some vessels. Increased egr-1 expression
has been associated with vascular occlusion, which could
very well lead to chylothorax (Merrigan et al., 1997;
de Beer et al., 2000). These potential links need to be
studied further in more detail. In summary, the netd/d mice
show that egr-1 is negatively regulated by the TCF Net
in vivo, and provide an interesting model to study the role
of Net in the regulation of egr-1 and more generally in
vascular biology.
Materials and methods
Generation of Net mutant mice
net gene sequences containing exon 2 were cloned from a 129Sv l
EMBL3 phage library and characterized by Southern blotting and
sequencing. The targeting sequences, containing, 5¢ to 3¢, a 11.5 kb
BamHI±AvaI fragment, a 1.8 kb PGK-neo cassette and a 1.2 kb
AvaI±BamHI fragment, were excised with NotI and electroporated into
D4 ES cells. Homologous recombinants were identi®ed by Southern
blotting and injected into C57BL/6 blastocysts to obtain chimeras. One of
the positive ES clones transmitted the disrupted allele. Pure 129Sv and
C57BL/6, and mixed (C57BL/6 3 129sv) genetic backgrounds were
used.
Genotyping of ES cells, embryos and mice
Genomic DNA from ES cells or tail biopsies was isolated and
resuspended in 100 ml of 10 mM Tris±HCl pH 8.0, 1 mM EDTA. A
15 ml aliquot of DNA was digested with XbaI and analysed by Southern
blotting using a 3.8 kb 3¢ probe from outside the targeting construct.
Routinely, mice were genotyped by PCR using the allele-speci®c primers:
UC54, TGAAACGTGTAATCCTTGTGTCCTC; UC56, TAATTTCCAAGTTCTCGGCACGTAG; and UC57, GACCGCTTCCTCGTGCTTTACGGTA.
PCR conditions were 94°C for 2 min, 30 cycles at 94°C for 30 s, 64°C
for 30 s, 72°C for 45 s and 72°C for 5 min, 0.5 ml of DNA, 200 ng of each
primer and 25 ml of reaction containing PCR reagents (Sigma). Fragments
of 1550 and 1300 bp are ampli®ed from the wild-type and targeted alleles,
respectively.
Expression analysis by RT±PCR and western blotting
RT±PCR. RNA was extracted from mouse tissues with Trizol (GibcoBRL). A 1 mg aliquot of total RNA was used for reverse transcription with
exon-speci®c primers and ampli®ed as described previously (Giovane
et al., 1994). The primer pairs were: EX1/EX2, CTAGAAATCTCCCCAAGAAGACTC/GTTGTCGTCATAGTATCTCAGCGC; EX2/EX3,
TGCTGGACATCGAACGATGGCGAG/ACTTGTACACAAACTTCTGCCCGA; EX3/EX4, CTGGAGCCCCTGAATCTGTCATCG/TCGAGGCCAGAAACAGTCCACTTG; and EX1/EX3, CTAGAAATCTCCCCAAGAAGACTC/ACTTGTACACAAACTTCTGCCCGA.
Net negatively regulates egr-1 in vivo
The PCR products were electrophoresed on 5% polyacrylamide gels,
stained with ethidium bromide and visualized under UV.
Western blots. Tissues were homogenized in RIPA buffer (150 mM NaCl,
1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris±HCl pH 8,
2 mg/ml aprotinin, 2 mg/ml leupeptin and 100 mg/ml phenylmethylsulfonyl ¯uoride) with an Ultrathorax. Proteins (200±300 mg) were
fractionated by 10% SDS±PAGE, transferred to nitrocellulose membranes and revealed with puri®ed PAb 375 (Giovane et al., 1994) and the
enhanced chemiluminescence detection kit (Amersham).
Histology, b-galactosidase staining and ISH
Standard conditions were used for histology, whole-mount b-galactosidase staining (Beddington et al., 1989) and ISH with 35S- and digoxigeninlabelled RNA probes (DeÂcimo et al., 1995). Probes were net (full-length
cDNA; Giovane et al., 1994), VEGF-R1 (RG 458440, Research Genetics
Inc.), VEGFR-3 (Kaipainen et al., 1995), egr-1 (nucleotides 744±1400)
and c-fos (DDBJ/EMBL/GenBank accession No. V00727). The yolk sacs
of embryos were used for PCR genotyping.
Cell culture and transfections
Mefs were isolated from wild-type and homozygous mutant embryos
(Robertson, 1987). Passage 3±6 Mefs were transfected in triplicate by the
DEAE±dextran method (al-Moslih and Dubes, 1973) in 6-well clusters
(Costar 3516) with 5 mg of DNA. After 20 h, the cells were washed twice
with Dulbecco's modi®ed Eagle's medium (DMEM), incubated in
DMEM containing 0.5% fetal calf serum (FCS) for 24 h, and scraped for
luciferase assays. NIH-3T3 cells in DMEM (Sigma) + 10% FCS were
transfected by the BBS calcium phosphate method in 6-well clusters with
4 mg of DNA per well containing 0.5 mg of the egr-1 reporters, 0.25±1 mg
of the antisense net plasmid (p601D-anti-Net) and the appropriate
amounts of the control vectors. After 16 h, the cells were washed,
incubated for 24 h and scraped for luciferase assays.
Mobility shift assays
pSG5-based expression vectors for Net, SRF and Netd were transcribed
and translated in TNT rabbit reticulocyte lysates (Promega). Proteins
(2 ml, adjusted to 4 ml with mock extracts that had been incubated with the
pSG5 backbone vector) and an excess of probes in 20 ml of buffer [20 mM
HEPES pH 7.8, 20% glycerol, 0.1 mM EDTA, 2.5 mM dithiothreitol
(DTT), 10 mg of poly(dI±dC), 100 mM KCl] were incubated for 30 min on
ice, 15 min at 37°C and 1 h at 25°C. The samples were loaded on pre-run
(45 min at 8 mA) 6.6% polyacrylamide gel in 0.253 TBE and run with
re-circulating buffer at 4°C.
The egr-1 SRE-5 probes were: egr-1 WT, 5¢-GTTCGCCGACCCGGAAACGCCATATAAGGAGCAGG-3¢; egr-1 mut ets, 5¢-GTTCGCCGACCCGCATATGCCATATAAGGAGCAGG-3¢; and egr-1 mut
SRF: 5¢-GTTCGCCGACCCGGAAACGCCATATGAAGAGCAGG-3¢.
One strand of the double-stranded blunt-end probes is shown. The Ets
and SRF motifs are underlined. Oligonucleotides were 5¢-end-labelled
with T4 polynucleotide kinase and puri®ed on native 10% polyacrylamide gels.
Plasmids
Plasmids were constructed by standard methods: pTL2-Net, pTL2-SRF,
p601D and p601D-anti-Net (Giovane et al., 1994); pKOZ1-Netd, the net
fragment from GAL-N1 in pKOZ1 (Maira et al., 1996); pGL2-egr-1200
and pGL2-egr-250 (Clarkson et al., 1999), kindly provided by Michael
J.Waters.
Acknowledgements
We would like to thank the IGBMC core facilities for help and support;
the Ligue ReÂgional (Bas-Rhin) contre le Cancer, the Association pour la
Recherche sur le Cancer and the MinisteÁre de la Recherche et de
Technologie for fellowships for A.A.; Aventis for a fellowship for H.Z.;
Human Frontiers for a fellowship for P.S.; and Aventis, the Centre
National de la Recherche Scienti®que, the Institut National de la Sante et
de la Recherche MeÂdicale, the HoÃpital Universitaire de Strasbourg, the
Association pour la Recherche sur le Cancer, the Fondation pour la
Recherche MeÂdicale, the Ligue Nationale FrancËaise contre le Cancer
(eÂquipe labelliseÂe), the Ligue ReÂgionale (Haut-Rhin) contre le Cancer and
the Ligue ReÂgionale (Bas-Rhin) contre le Cancer for ®nancial help.
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Received December 8, 2000; revised July 6, 2001;
accepted July 24, 2001